Compounds for eliciting or enhancing immune reactivity to HER-2/neu protein for prevention or treatment of malignancies in which the HER-2/neu oncogene is associated

ABSTRACT

Compounds and compositions for eliciting or enhancing immune reactivity to HER-2/neu protein are disclosed. The compounds include polypeptides and nucleic acid molecules encoding such peptides. The compounds may be used for the prevention or treatment of malignancies in which the HER-2/neu oncogene is associated.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part to Ser. No. 414,417, filedMar. 31, 1995, which is a continuation-in-part application to Ser. No.106,112, filed Aug. 12, 1993, abandoned, which is a continuation-in-partapplication to Ser. No. 033,644, filed Mar. 17, 1993, abandoned.

TECHNICAL FIELD

The present invention is generally directed toward polypeptides, andnucleic acid molecules encoding such polypeptides, for eliciting orenhancing an immune response to HER-2/neu protein, including for use inthe treatment of malignancies in which the HER-2/neu oncogene isassociated.

BACKGROUND OF THE INVENTION

Despite enormous investments of financial and human resources, cancerremains one of the major causes of death. For example, cancer is theleading cause of death in women between the ages of 35 and 74. Breastcancer is the most common malignancy in women and the incidence fordeveloping breast cancer is on the rise. One in nine women will bediagnosed with the disease. Standard approaches to cure breast cancerhave centered around a combination of surgery, radiation andchemotherapy. These approaches have resulted in some dramatic successesin certain malignancies. However, these approaches have not beensuccessful for all malignancies and breast cancer is most oftenincurable when attempting to treat beyond a certain stage. Alternativeapproaches to prevention and therapy are necessary.

A common characteristic of malignancies is uncontrolled cell growth.Cancer cells appear to have undergone a process of transformation fromthe normal phenotype to a malignant phenotype capable of autonomousgrowth. Amplification and overexpression of somatic cell genes isconsidered to be a common primary event that results in thetransformation of normal cells to malignant cells. The malignantphenotypic characteristics encoded by the oncogenic genes are passed onduring cell division to the progeny of the transformed cells.

Ongoing research involving oncogenes has identified at least fortyoncogenes operative in malignant cells and responsible for, orassociated with, transformation. Oncogenes have been classified intodifferent groups based on the putative function or location of theirgene products (such as the protein expressed by the oncogene).

Oncogenes are believed to be essential for certain aspects of normalcellular physiology. In this regard, the HER-2/neu oncogene is a memberof the tyrosine protein kinase family of oncogenes and shares a highdegree of homology with the epidermal growth factor receptor. HER-2/neupresumably plays a role in cell growth and/or differentiation. HER-2/neuappears to induce malignancies through quantitative mechanisms thatresult from increased or deregulated expression of an essentially normalgene product.

HER-2/neu (p185) is the protein product of the HER-2/neu oncogene. TheHER-2/neu gene is amplified and the HER-2/neu protein is overexpressedin a variety of cancers including breast, ovarian, colon, lung andprostate cancer. HER-2/neu is related to malignant transformation. It isfound in 50%-60% of ductal in situ carcinoma and 20%-40% of all breastcancers, as well as a substantial fraction of adenocarcinomas arising inthe ovaries, prostate, colon and lung. HER-2/neu is intimatelyassociated not only with the malignant phenotype, but also with theaggressiveness of the malignancy, being found in one-fourth of allinvasive breast cancers. HER-2/neu overexpression is correlated with apoor prognosis in both breast and ovarian cancer. HER-2/neu is atransmembrane protein with a relative molecular mass of 185 kd that isapproximately 1255 amino acids (aa) in length. It has an extracellularbinding domain (ECD) of approximately 645 aa, with 40% homology toepidermal growth factor receptor (EGFR), a highly hydrophobictransmembrane anchor domain (TMD), and a carboxyterminal cytoplasmicdomain (CD) of approximately 580 aa with 80% homology to EGFR.

Due to the difficulties in the current approaches to therapy of cancersin which the HER-2/neu oncogene is associated, there is a need in theart for improved compounds and compositions. The present inventionfulfills this need, and further provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides polypeptides, nucleicacid molecules (directing the expression of such polypeptides) and viralvectors (directing the expression of such polypeptides) for uses whichinclude the immunization of a warm-blooded animal against a malignancyin which the HER-2/neu oncogene is associated. A polypeptide or nucleicacid molecule according to this invention may be present in acomposition that includes a pharmaceutically acceptable carrier ordiluent. Such a polypeptide, nucleic acid molecule, viral vector orpharmaceutical composition may be administered on a one-time basis(e.g., when a malignancy is suspected) or on a periodic basis (e.g., foran individual with an elevated risk of acquiring or reacquiring amalignancy). A compound or composition of the present invention may beuseful in the treatment of an existing tumor or to prevent tumoroccurrence or reoccurrence.

In one aspect, the present invention provides compounds and compositionsthat elicit or enhance an immune response to HER-2/neu protein. Oneembodiment of the present invention provides a polypeptide encoded by aDNA sequence selected from: (a) nucleotides 2026 through 3765 of SEQ IDNO:1; and (b) DNA sequences that hybridize to a nucleotide sequencecomplementary to nucleotides 2026 through 3765 of SEQ ID NO:1 undermoderately stringent conditions, wherein the DNA sequence encodes apolypeptide that produces an immune response to HER-2/neu protein. In apreferred embodiment, a polypeptide has the amino acid sequence of SEQID NO:2 from lysine, amino acid 676, through valine, amino acid 1255, ora variant thereof that produces at least an equivalent immune response.A composition is provided that comprises a polypeptide of the presentinvention in combination with a pharmaceutically acceptable carrier ordiluent. In another embodiment, a nucleic acid molecule directing theexpression of a polypeptide according to the present invention isprovided. In another embodiment, a viral vector directing the expressionof a polypeptide according to the present invention is provided.

In another aspect, the present invention provides a method for elicitingor enhancing an immune response to HER-2/neu protein, comprisingadministering to a warm-blooded animal (such as a human) in an amounteffective to elicit or enhance the response a polypeptide according tothe present invention, or a nucleic acid molecule or a viral vector,either directing the expression of such a polypeptide. In oneembodiment, a peptide is administered in combination with apharmaceutically acceptable carrier or diluent. In another embodiment,the step of administering comprises transfecting cells of the animal exvivo with the nucleic acid molecule and subsequently delivering thetransfected cells to the animal. In another embodiment, the step ofadministering comprises infecting cells of the animal ex vivo with theviral vector and subsequently delivering the infected cells to theanimal.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of the priming of naive T lymphocytes toHER-2/neu polypeptide by dendritic cells. Bone marrow-derived DC weregenerated with GM-CSF and IL6 from CD34+ stem cells. DC pulsed withHER-2/neu polypeptide induced protein-specific proliferation OLautologous CD4+/CD45RA+ T lymphocytes after 7 days of culturing T cellswith DC. Bone marrow-derived CD34+ stem cells cultured for ore week inserum-free medium containing GM-CSF and IL-6 were used as APC. APC wereplated into 96-well round-bottomed plates (Corning, Corning, N.Y., USA)at various concentrations and incubated for 16-18 hours with 20-25 μg/mlof recombinant HER-2/neu polypeptide. CD4+ T lymphocytes were isolatedfrom autologous peripheral blood mononuclear cells by positive selectionusing immunoaffinity columns (CellPro, Inc., Bothell, Wash., USA).Antigen-pulsed APC were irradiated (10 Gy), and CD4+ T lymphocytes wereadded at 10⁵ per well. Proliferative response of T cells was measured bythe uptake of (³H) thymidine (1 μCi/well) added on day 7 for 16-18hours. Proliferation assays were performed in serum- and cytokine-freemedium in 5 well replicates. The symbols represent: —●— DC+HER-2/neupolypeptide+CD4+/CD45RA+ T cells; —◯— DC+CD4+/CD45RA+ T cells; and -□-DC+HER-2/neu polypeptide.

FIG. 2 shows the response of CD4+ cells to HER-2/neu polypeptide. Usingthe priming assay described for FIG. 1, CD4+ T cells from normal donorswere tested for responses to recombinant human HER-2/neu polypeptide.The symbols represent: —●— SC+CD4; and ••••••••♦••••••••SC+CD4+HER-2/neu polypeptide. “SC” is stem cells.

FIG. 3 shows that rats immunized with rat HER-2/neu polypeptide developrat neu specific antibodies. Rats were immunized with recombinant ratHER-2/neu polypeptide 25 ug in MPL or vaccel adjuvant. Threeimmunizations were given, each 20 days apart. Twenty days after thefinal immunization rats were assessed for antibody responses to rat neu.Animals immunized with rat HER-2/neu polypeptide and the vaccel adjuvantshowed high titer rat neu specific responses. The control was an animalimmunized with human HER-2/neu polypeptide (foreign protein). Inseparate experiments, rats immunized with 100 ug and 300 ug of purifiedwhole rat neu did not develop detectable neu specific antibodies (datanot shown). Data represents the mean and standard deviation of 3animals. The symbols represent: —▪— rat HER-2/neu polypeptide/MPL;•••••••●••••••• rat HER-2/neu polypeptide/vaccel; ----□---- MPL alone;----◯---- vaccel alone; and

control. “MPL” and “vaccel” are adjuvants (Ribi, Bozeman, Mont., USA)“Neu” is HER-2/neu protein.

FIG. 4 shows that breast cancer patients have preexistent immunity toHER-2/neu polypeptide. Patient PBMC were evaluated by tritiatedthymidine incorporation in 24 well replicates. Responsive wells arescored as greater than the mean and 3 standard deviations (372 cpm) ofthe control wells. This HER-2/neu positive-stage II breast cancerpatient has a significant response to recombinant human HER-2/neupolypeptide. The symbols “p” represent peptides for HER-2/neu protein,“tt” represents tetanus toxoid, and “hHNP” represents recombinant humanHER-2/neu polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms to beused hereinafter.

HER-2/neu polypeptide—as used herein, refers to a portion of theHER-2/neu protein (the protein also known as p185 or c-erbB2) having theamino acid sequence of SEQ ID NO:2 from lysine, amino acid 676, throughvaline, amino acid 1255; and may be naturally derived, syntheticallyproduced, genetically engineered, or a functionally equivalent variantthereof, e.g., where one or more amino acids are replaced by other aminoacid(s) or non-amino acid(s) which do not substantially affectelicitation or enhancement of an immune response to HER-2/neu protein(e.g., variant stimulates a response by helper T cells or cytotoxic Tcells).

Proliferation of T cells—as used herein, includes the multiplication ofT cells as well as the stimulation of T cells leading to multiplication,i.e., the initiation of events leading to mitosis and mitosis itself.Methods for detecting proliferation of T cells are discussed below.

As noted above, the present invention is directed toward compounds andcompositions to elicit or enhance immunity to the protein productexpressed by the HER-2/neu oncogene, including for malignancies in awarm-blooded animal wherein an amplified HER-2/neu gene is associatedwith the malignancies. Association of an amplified HER-2/neu gene with amalignancy does not require that the protein expression product of thegene be present on the tumor. For example, overexpression of the proteinexpression product may be involved with initiation of a tumor, but theprotein expression may subsequently be lost. A use of the presentinvention is to elicit or enhance an effective autochthonous immuneresponse to convert a HER-2/neu positive tumor to HER-2/neu negative.

More specifically, the disclosure of the present invention, in oneaspect, shows that a polypeptide based on a particular portion(HER-2/neu polypeptide) of the protein expression product of theHER-2/neu gene can be recognized by thymus-dependent lymphocytes(hereinafter “T cells”) and, therefore, the autochthonous immune T cellresponse can be utilized prophylactically or to treat malignancies inwhich such a protein is or has been overexpressed. The disclosure of thepresent invention also shows, in another aspect, that nucleic acidmolecules directing the expression of such a peptide may be used aloneor in a viral vector for immunization.

In general, CD4+ T cell populations are considered to function ashelpers/inducers through the release of lymphokines when stimulated by aspecific antigen; however, a subset of CD4⁺ cells can act as cytotoxic Tlymphocytes (CTL). Similarly, CD8⁺ T cells are considered to function bydirectly lysing antigenic targets; however, under a variety ofcircumstances they can secrete lymphokines to provide helper or DTHfunction. Despite the potential of overlapping function, the phenotypicCD4 and CD8 markers are linked to the recognition of peptides bound toclass II or class I MHC antigens. The recognition of antigen in thecontext of class II or class I MHC mandates that CD4⁺ and CD8⁺ T cellsrespond to different antigens or the same antigen presented underdifferent circumstances. The binding of immunogenic peptides to class IIMHC antigens most commonly occurs for antigens ingested by antigenpresenting cells. Therefore, CD4⁺ T cells generally recognize antigensthat have been external to the tumor cells. By contrast, under normalcircumstances, binding of peptides to class I MHC occurs only forproteins present in the cytosol and synthesized by the target itself,proteins in the external environment are excluded. An exception to thisis the binding of exogenous peptides with a precise class I bindingmotif which are present outside the cell in high concentration. Thus,CD4⁺ and CD8⁺ T cells have broadly different functions and tend torecognize different antigens as a reflection of where the antigensnormally reside.

As disclosed within the present invention, a polypeptide portion of theprotein product expressed by the HER-2/neu oncogene is recognized by Tcells. Circulating HER-2/neu polypeptide is degraded to peptidefragments. Peptide fragments from the polypeptide bind to majorhistocompatibility complex (MHC) antigens. By display of a peptide boundto MHC antigen on the cell surface and recognition by host T cells ofthe combination of peptide plus self MHC antigen, HER-2/neu polypeptide(including that expressed on a malignant cell) will be immunogenic to Tcells. The exquisite specificity of the T cell receptor enablesindividual T cells to discriminate between peptides which differ by asingle amino acid residue.

During the immune response to a peptide fragment from the polypeptide, Tcells expressing a T cell receptor with high affinity binding of thepeptide-MHC complex will bind to the peptide-MHC complex and therebybecome activated and induced to proliferate. In the first encounter witha peptide, small numbers of immune T cells will secrete lymphokines,proliferate and differentiate into effector and memory T cells. Theprimary immune response will occur in vivo but has been difficult todetect in vitro. Subsequent encounter with the same antigen by thememory T cell will lead to a faster and more intense immune response.The secondary response will occur either in vivo or in vitro. The invitro response is easily gauged by measuring the degree ofproliferation, the degree of cytokine production, or the generation ofcytolytic activity of the T cell population re-exposed in the antigen.Substantial proliferation of the T cell population in response to aparticular antigen is considered to be indicative of prior exposure orpriming to the antigen.

The compounds of this invention generally comprise HER-2/neupolypeptides or DNA molecules that direct the expression of suchpeptides, wherein the DNA molecules may be present in a viral vector. Asnoted above, the polypeptides of the present invention include variantsof the polypeptide of SEQ ID NO:2 from amino acid 676 through amino acid1255, that retain the ability to stimulate an immune response. Suchvariants include various structural forms of the native polypeptide. Dueto the presence of ionizable amino and carboxyl groups, for example, aHER-2/neu polypeptide may be in the form of an acidic or basic salt, ormay be in neutral form. Individual amino acid residues may also bemodified by oxidation or reduction.

Variants within the scope of this invention also include polypeptides inwhich the primary amino acid structure native HER-2/neu polypeptide ismodified by forming covalent or aggregative conjugates with otherpeptides or polypeptides, or chemical moieties such as glycosyl groups,lipids, phosphate, acetyl groups and the like. Covalent derivatives maybe prepared, for example, by linking particular functional groups toamino acid side chains or at the N- or C-terminus.

The present invention also includes HER-2/neu polypeptides with orwithout glycosylation. Polypeptides expressed in yeast or mammalianexpression systems may be similar to or slightly different in molecularweight and glycosylation pattern than the native molecules, dependingupon the expression system. For instance, expression of DNA encodingpolypeptides in bacteria such as E. coli typically providesnon-glycosylated molecules. N-glycosylation sites of eukaryotic proteinsare characterized by the amino acid triplet Asn-A₁-Z, where A₁ is anyamino acid except Pro, and Z is Ser or Thr. Variants of HER-2/neupolypeptides having inactivated N-glycosylation sites can be produced bytechniques known to those of ordinary skill in the art, such asoligonucleotide synthesis and ligation or site-specific mutagenesistechniques, and are within the scope of this invention. Alternatively,N-linked glycosylation sites can be added to a HER-2/neu polypeptide.

The polypeptides of this invention also include variants of the SEQ IDNO:2 polypeptide (i.e., variants of a polypeptide having the amino acidsequence of SEQ ID NO:2 from amino acid 676 through amino acid 1255)that have an amino acid sequence different from this sequence because ofone or more deletions, insertions, substitutions or other modifications.In one embodiment, such variants are substantially homologous to thenative HER-2/neu polypeptide and retain the ability to stimulate animmune response. “Substantial homology,” as used herein, refers to aminoacid sequences that may be encoded by DNA sequences that are capable ofhybridizing under moderately stringent conditions to a nucleotidesequence complimentary to a naturally occurring DNA sequence encodingthe specified polypeptide portion of SEQ ID NO:2 herein (i.e.,nucleotides 2026 through 3765 of SEQ ID NO:1). Suitable moderatelystringent conditions include prewashing in a solution of −5×SSC, 0.5%SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50° C.-65° C., 5×SSC,overnight; followed by washing twice at 65° C. for 20 minutes with eachof 2×, 0.5× and 0.2×SSC (containing 0.1% SDS). Such hybridizing DNAsequences are also within the scope of this invention. The effect of anysuch modifications on the ability of a HER-2/neu polypeptide to producean immune response may be readily determined (e.g., by analyzing theability of the mutated HER-2/neu polypeptide to induce a T cell responseusing, for example, the methods described herein).

Generally, amino acid substitutions may be made in a variety of ways toprovide other embodiments of variants within the present invention.First, for example, amino acid substitutions may be made conservatively;i.e., a substitute amino acid replaces an amino acid that has similarproperties, such that one skilled in the art of peptide chemistry wouldexpect the secondary structure and hydropathic nature of the polypeptideto be substantially unchanged. In general, the following groups of aminoacids represent conservative changes: (1) ala, pro, gly, glu, asp, gin,asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe;(4) lys, arg, his; and (5) phe, tyr, trp, his. An example of anon-conservative change is to replace an amino acid of one group with anamino acid from another group.

Another way to make amino acid substitutions to produce variants of thepresent invention is to identify and replace amino acids in T cellmotifs with potential to bind to class II MHC molecules (for CD4+ T cellresponse) or class I MHC molecules (for CD8+ T cell response) Peptidesegments (of a HER-2/neu polypeptide) with a motif with theoreticalpotential to bind to class II MHC molecules may be identified bycomputer analysis. For example, a protein sequence analysis package, TSites, that incorporates several computer algorithms designed todistinguish potential sites for T cell recognition can be used (Fellerand de la Cruz, Nature 349:720-721, 1991). Two searching algorithms areused: (1) the AMPHI algorithm described by Margalit (Feller and de laCruz, Nature 349:720-721, 1991; Margalit et al., J. Immunol.138:2213-2229, 1987) identifies epitope motifs according toalpha-helical periodicity and amphipathicity; (2) the Rothbard andTaylor algorithm identifies epitope motifs according to charge andpolarity pattern (Rothbard and Taylor, EMBO 7:93-100, 1988). Segmentswith both motifs are most appropriate for binding to class II MHCmolecules. CD8⁺ T cells recognize peptide bound to class I MHCmolecules. Falk et al. have determined that peptides binding toparticular MHC molecules share discernible sequence motifs (Falk et al.,Nature 351:290-296, 1991). A peptide motif for binding in the groove ofHLA-A2.1 has been defined by Edman degradation of peptides stripped fromHLA-A2.1 molecules of a cultured cell line (Table 2, from Falk et al.,supra). The method identified the typical or average HLA-A2.1 bindingpeptide as being 9 amino acids in length with dominant anchor residuesoccurring at positions 2 (L) and 9 (V). Commonly occurring strongbinding residues have been identified at positions 2 (M), 4 (E,K), 6(V), and 8 (K). The identified motif represents the average of manybinding peptides. The HLA-A2.1 Restricted Motif Amino Acid PositionPoint 1 2 3 4 5 6 7 8 9 Assignment Dominant Binding   L             V +3Anchor Residue Strong Binding   M   E   V   K +2 Residue       K WeakBinding I   A G I I A E L +1 Residue L   Y P K L Y S F   F D Y T HK   P T N M   M   G Y   S   V         HThe derived peptide motif as currently defined is not particularlystringent. Some HLA-A2.1 binding peptides do not contain both dominantanchor residues and the amino acids flanking the dominant anchorresidues play major roles in allowing or disallowing binding. Not everypeptide with the current described binding motif will bind, and somepeptides without the motif will bind. However, the current motif isvalid enough to allow identification of some peptides capable ofbinding. Of note, the current HLA-A2.1 motif places 6 amino acidsbetween the dominant anchor amino acids at residues 2 and 9.

Following identification of peptide motifs within a HER-2/neupolypeptide, amino acid substitutions may be made conservatively ornon-conservatively. The latter type of substitutions are intended toproduce an improved polypeptide that is more potent and/or more broadlycross-reactive (MHC polymorphism). An example of a more potentpolypeptide is one that binds with higher affinity to the same MHCmolecule as natural polypeptide, without affecting recognition by Tcells specific for natural polypeptide. An example of a polypeptide withbroader cross-reactivity is one that induces more broadly cross-reactiveimmune responses (i.e., binds to a greater range of MHC molecules) thannatural polypeptide. Similarly, one or more amino acids residing betweenpeptide motifs and having a spacer function (e.g., do not interact witha MHC molecule or T cell receptor) may be substituted conservatively ornon-conservatively. It will be evident to those of ordinary skill in theart that polypeptides containing one or more amino acid substitutionsmay be tested for beneficial or adverse immunological interactions by avariety of assays, including those described herein for the ability tostimulate T cell recognition.

Variants within the scope of this invention may also, or alternatively,contain other modifications, including the deletion or addition of aminoacids, that have minimal influence on the desired immunologicalproperties of the polypeptide. It will be appreciated by those ofordinary skill in the art that truncated forms or non-native extendedforms of a HER-2/neu polypeptide may be used, provided the desiredimmunological properties are at least roughly equivalent to that of fulllength, native HER-2/neu polypeptide. Cysteine residues may be deletedor replaced with other amino acids to prevent formation of incorrectintramolecular disulfide bridges upon renaturation. Other approaches tomutagenesis involve modification of adjacent dibasic amino acid residuesto enhance expression in yeast systems in which KEX2 protease activityis present.

A HER-2/neu polypeptide may generally be obtained using a genomic orcDNA clone encoding the protein. A genomic sequence that encodes fulllength HER-2/neu is shown in SEQ ID NO:1, and the deduced amino acidsequence is presented in SEQ ID NO:2. Such clones may be isolated byscreening an appropriate expression library for clones that expressHER-2/neu protein. The library preparation and screen may generally beperformed using methods known to those of ordinary skill in the art,such as methods described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor,N.Y., 1989, which is incorporated herein by reference. Briefly, abacteriophage expression library may be plated and transferred tofilters. The filters may then be incubated with a detection reagent. Inthe context of this invention, a “detection reagent” is any compoundcapable of binding to HER-2/neu protein, which may then be detected byany of a variety of means known to those of ordinary skill in the art.Typical detection reagents contain a “binding agent,” such as Protein A,Protein G, IgG or a lectin, coupled to a reporter group. Preferredreporter groups include enzymes, substrates, cofactors, inhibitors,dyes, radionuclides, luminescent groups, fluorescent groups and biotin.More preferably, the reporter group is horseradish peroxidase, which maybe detected by incubation with a substrate such as tetramethylbenzidineor 2,2′-azino-di-3-ethylbenzthiazoline sulfonic acid. Plaques containinggenomic or cDNA sequences that express HER-2/neu protein are isolatedand purified by techniques known to those of ordinary skill in the art.Appropriate methods may be found, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories,Cold Spring Harbor, N.Y., 1989.

Variants of the polypeptide that retain the ability to stimulate animmune response may generally be identified by modifying the sequence inone or more of the aspects described above and assaying the resultingpolypeptide for the ability to stimulate an immune response, e.g., a Tcell response. For example, such assays may generally be performed bycontacting T cells with the modified polypeptide and assaying theresponse. Naturally occurring variants of the polypeptide may also beisolated by, for example, screening an appropriate cDNA or genomiclibrary with a DNA sequence encoding the polypeptide or a variantthereof.

The above-described sequence modifications may be introduced usingstandard recombinant techniques or by automated synthesis of themodified polypeptide. For example, mutations can be introduced atparticular loci by synthesizing oligonucleotides containing a mutantsequence, flanked by restriction sites enabling ligation to fragments ofthe native sequence. Following ligation, the resulting reconstructedsequence encodes an analogue having the desired amino acid insertion,substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide a gene in which particular codonsare altered according to the substitution, deletion, or insertionrequired. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al., Gene 42:133, 1986; Bauer et al., Gene37:73, 1985; Craik, BioTechniques, January 1985, 12-19; Smith et al.,Genetic Engineering: Principles and Methods, Plenum Press, 1981; andU.S. Pat. Nos. 4,518,584 and 4,737,462.

Mutations in nucleotide sequences constructed for expression of suchHER-2/neu polypeptides must, of course, preserve the reading frame ofthe coding sequences and preferably will not create complementaryregions that could hybridize to produce secondary mRNA structures, suchas loops or hairpins, which would adversely affect translation of themRNA. Although a mutation site may be predetermined, it is not necessarythat the nature of the mutation per se be predetermined. For example, inorder to select for optimum characteristics of mutants at a given site,random mutagenesis may be conducted at the target codon and theexpressed HER-2/neu polypeptide mutants screened for the desiredactivity.

Not all mutations in a nucleotide sequence which encodes a HER-2/neupolypeptide will be expressed in the final product. For example,nucleotide substitutions may be made to enhance expression, primarily toavoid secondary structure loops in the transcribed mRNA (see, e.g.,European Patent Application 75,444A), or to provide codons that are morereadily translated by the selected host, such as the well-known E. colipreference codons for E. coli expression.

The polypeptides of the present invention, both naturally occurring andmodified, are preferably produced by recombinant DNA methods. Suchmethods include inserting a DNA sequence encoding a HER-2/neupolypeptide into a recombinant expression vector and expressing the DNAsequence in a recombinant microbial, mammalian or insect cell expressionsystem under conditions promoting expression. DNA sequences encoding thepolypeptides provided by this invention can be assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene which is capable of beinginserted in a recombinant expression vector and expressed in arecombinant transcriptional unit.

Recombinant expression vectors contain a DNA sequence encoding aHER-2/neu polypeptide operably linked to suitable transcriptional ortranslational regulatory elements derived from mammalian, microbial,viral or insect genes. Such regulatory elements include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences which control the termination of transcription andtranslation. An origin of replication and a selectable marker tofacilitate recognition of transformants may additionally beincorporated.

DNA regions are operably linked when they are functionally related toeach other. For example, DNA for a signal peptide (secretory leader) isoperably linked to DNA for a polypeptide if it is expressed as aprecursor which participates in the secretion of the polypeptide; apromoter is operably linked to a coding sequence if it controls thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to permittranslation. Generally, operably linked means contiguous and, in thecase of secretory leaders, in reading frame. DNA sequences encodingHER-2/neu polypeptides which are to be expressed in a microorganism willpreferably contain no introns that could prematurely terminatetranscription of DNA into mRNA.

Expression vectors for bacterial use may comprise a selectable markerand bacterial origin of replication derived from commercially availableplasmids comprising genetic elements of the well known cloning vectorpBR322 (ATCC 37017). Such commercial vectors include, for example,pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (PromegaBiotec, Madison, Wis., USA). These pBR322 “backbone” sections arecombined with an appropriate promoter and the structural sequence to beexpressed. E. coli is typically transformed using derivatives of pBR322,a plasmid derived from an E. coli species (Bolivar et al., Gene 2:95,1977). pBR322 contains genes for ampicillin and tetracycline resistanceand thus provides simple means for identifying transformed cells.

Promoters commonly used in recombinant microbial expression vectorsinclude the β-lactamase (penicillinase) and lactose promoter system(Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544,1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. AcidsRes. 8:4057, 1980; and European Patent Application 36,776) and the tacpromoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, p. 412, 1982). A particularly useful bacterialexpression system employs the phage λ P_(L) promoter and cI857tsthermolabile repressor. Plasmid vectors available from the American TypeCulture Collection which incorporate derivatives of the λ P_(L) promoterinclude plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) andpPLc28, resident in E. coli RR1 (ATCC 53082).

Suitable promoter sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Suitable vectorsand promoters for use in yeast expression are further described in R.Hitzeman et al., European Patent Application 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pBR322for selection and replication in E. coli (Amp^(r) gene and origin ofreplication) and yeast DNA sequences including a glucose-repressibleADH2 promoter and α-factor secretion leader. The ADH2 promoter has beendescribed by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier etal. (Nature. 300:724, 1982). The yeast α-factor leader, which directssecretion of heteroldgous proteins, can be inserted between the promoterand the structural gene to be expressed (see, e.g., Kurjan et al., Cell30:933, 1982; and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330,1984). The leader sequence may be modified to contain, near its 31′ end,one or more useful restriction sites to facilitate fusion of the leadersequence to foreign genes. The transcriptional and translational controlsequences in expression vectors to be used in transforming vertebratecells may be provided by viral sources. For example, commonly usedpromoters and enhancers are derived from polyoma, adenovirus 2, simianvirus 40 (SV40), and human cytomegalovirus. DNA sequences derived fromthe SV40 viral genome, for example, SV40 origin, early and latepromoter, enhancer, splice, and polyadenylation sites may be used toprovide the other genetic elements required for expression of aheterologous DNA sequence. The early and late promoters are particularlyuseful because both are obtained easily from the virus as a fragmentwhich also contains the SV40 viral origin of replication (Fiers et al.,Nature 273:113, 1978). Smaller or larger SV40 fragments may also beused, provided the approximately 250 bp sequence extending from the HindIII site toward the Bgl II site located in the viral origin ofreplication is included. Further, Viral genomic promoter, control and/orsignal sequences may be utilized, provided such control sequences arecompatible with the host cell chosen. Exemplary vectors can beconstructed as disclosed by Okayama and Berg, Mol. Cell. Biol. 3:280,1983.

A useful system for stable high level expression of mammalian receptorcDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986). A preferred eukaryotic vector for expression of HER-2/neupolypeptide DNA is pDC406 (McMahan et al., EMBO J. 10:2821, 1991), andincludes regulatory sequences derived from SV40, human immunodeficiencyvirus (HIV), and Epstein-Barr virus (EBV). Other preferred vectorsinclude pDC409 and pDC410, which are derived from pDC406. pDC410 wasderived from pDC406 by substituting the EBV origin of replication withsequences encoding the SV40 large T antigen. pDC409 differs from pDC406in that a Bgl II restriction site outside of the multiple cloning sitehas been deleted, making the Bgl II site within the multiple cloningsite unique.

A useful cell line that allows for episomal replication of expressionvectors, such as pDC406 and pDC409, which contain the EBV origin ofreplication, is CV-1/EBNA (ATCC CRL 10478). The CV-L/EBNA cell line wasderived by transfection of the CV-1 cell line with a gene encodingEpstein-Barr virus nuclear antigen-I (EBNA-1) and constitutively expressEBNA-1 driven from human CMV immediate-early enhancer/promoter.

Transformed host cells are cells which have been transformed ortransfected with expression vectors constructed using recombinant DNAtechniques and which contain sequences encoding a HER-2/neu polypeptideof the present invention. Transformed host cells may express the desiredHER-2/neu polypeptide, but host cells transformed for purposes ofcloning or amplifying HER-2/neu DNA do not need to express the HER-2/neupolypeptide. Expressed polypeptides will preferably be secreted into theculture supernatant, depending on the DNA selected, but may also bedeposited in the cell membrane.

Suitable host cells for expression of recombinant proteins includeprokaryotes, yeast or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or Bacilli. Higher eukaryoticcells include established cell lines of insect or mammalian origin asdescribed below. Cell-free translation systems could also be employed toproduce HER-2/neu polypeptides using RNAs derived from DNA constructs.Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are described, for example,by Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, N.Y.,1985.

Prokaryotic expression hosts may be used for expression of HER-2/neupolypeptides that do not require extensive proteolytic and disulfideprocessing. Prokaryotic expression vectors generally comprise one or orephenotypic selectable markers, for example a gene encoding proteinsconferring antibiotic resistance or supplying an autotrophicrequirement, and an origin of replication recognized by the host toensure amplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium, and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although other hosts may also beemployed.

Recombinant HER-2/neu polypeptides may also be expressed in yeast hosts,preferably from the Saccharomyces species, such as S. cerevisiae. Yeastof other genera, such as Pichia or Kluyveromyces may also be employed.Yeast vectors will generally contain an origin of replication from the2μ yeast plasmid or an autonomously replicating sequence (ARS), apromoter, DNA encoding the HER-2/neu polypeptide, sequences forpolyadenylation and transcription termination and a selection gene.Preferably, yeast vectors will include an origin of replication andselectable marker permitting transformation of both yeast and E. coli,e.g., the ampicillin resistance gene of E. coli and the S. cerevisiaetrp1 gene, which provides a selection marker for a mutant strain ofyeast lacking the ability to grow in tryptophan, and a promoter derivedfrom a highly expressed yeast gene to induce transcription of astructural sequence downstream. The presence of the trp1 lesion in theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.

Suitable yeast transformation protocols are known to those of skill inthe art. An exemplary technique described by Hind et al. (Proc. Natl.Acad. Sci. USA 75:1929, 1978), involves selecting for Trp⁺ transformantsin a selective medium consisting of 0.67% yeast nitrogen base, 0.5%casamino acids, 2% glucose, 10 mg/ml adenine and 20 mg/ml uracil. Hoststrains transformed by vectors comprising the ADH2 promoter may be grownfor expression in a rich medium consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 mg/ml adenine and 80 mg/mluracil. Derepression of the ADH2 promoter occurs upon exhaustion ofmedium glucose. Crude yeast supernatants are harvested by filtration andheld at 4° C. prior to further purification.

Various mammalian or insect (e.g., Spodoptera or Trichoplusia) cellculture systems can also be employed to express recombinant polypeptide.Baculovirus systems for production of heterologous polypeptides ininsect cells are reviewed, for example, by Luckow and Summers,Bio/Technology 6:47, 1988. Examples of suitable mammalian host celllines include the COS-7 lines of monkey kidney cells, described byGluzman (Cell 23:175, 1981), and other cell lines capable of expressingan appropriate vector including, for example, CV-1/EBNA (ATCC CRL10478), L cells, C127, 3T3, Chinese hamster ovary (CHO), COS, NS-1, HeLaand BHK cell lines. Mammalian expression vectors may comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 31 flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences.

Purified HER-2/neu polypeptides may be prepared by culturing suitablehost/vector systems to express the recombinant translation products ofthe DNAs of the present invention, which are then purified from culturemedia or cell extracts. For example, supernatants from systems whichsecrete recombinant polypeptide into culture media may be firstconcentrated using a commercially available protein concentrationfilter, such as an Amicon or Millipore Pellicon ultrafiltration unit.Following the concentration step, the concentrate may be applied to asuitable purification matrix. For example, a suitable affinity matrixmay comprise a counter structure protein (i.e., a protein to which aHER-2/neu polypeptide binds in a specific interaction based onstructure) or lectin or antibody molecule bound to a suitable support.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are preferred. Gel filtration chromatographyalso provides a means of purifying a HER-2/neu.

Affinity chromatography is a preferred method of purifying HER-2/neupolypeptides. For example, monoclonal antibodies against the HER-2/neupolypeptide may also be useful in affinity chromatography purification,by utilizing methods that are well-known in the art.

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media(e.g., silica gel having pendant methyl or other aliphatic groups) maybe employed to further purify a HER-2/neu polypeptide composition. Someor all of the foregoing purification steps, in various combinations, canalso be employed to provide a homogeneous recombinant polypeptide.

Recombinant HER-2/neu polypeptide produced in bacterial culture ispreferably isolated by initial extraction from cell pellets, followed byone or more concentration, salting-out, aqueous ion exchange or sizeexclusion chromatography steps. High performance liquid chromatography(HPLC) may be employed for final purification steps. Microbial cellsemployed in expression of recombinant HER-2/neu polypeptide can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents.

Fermentation of yeast which express HER-2/neu polypeptide as a secretedprotein greatly simplifies purification. Secreted recombinant proteinresulting from a large-scale fermentation can be purified by methodsanalogous to those disclosed by Urdal et al. (J. Chromatog. 296:171,1984). This reference describes two sequential, reverse-phase HPLC stepsfor purification of recombinant human GM-CSF on a preparative HPLCcolumn.

Preparations of HER-2/neu polypeptides synthesized in recombinantculture may contain non-HER-2/neu cell components, including proteins,in amounts and of a character which depend upon the purification stepstaken to recover the HER-2/neu polypeptide from the culture. Thesecomponents ordinarily will be of yeast, prokaryotic or non-humaneukaryotic origin. Such preparations are typically free of otherproteins which may be normally associated with the HER-2/neu protein asit is found in nature in its species of origin.

Automated synthesis provides an alternate method for preparingpolypeptides of this invention. For example, any of the commerciallyavailable solid-phase techniques may be employed, such as the Merrifieldsolid phase synthesis method, in which amino acids are sequentiallyadded to a growing amino acid chain. (See Merrifield, J. Am. Chem. Soc.85:2149-2146, 1963.) Equipment for automated synthesis of polypeptidesis commercially available from suppliers such as Applied Biosystems,Inc. of Foster City, Calif., and may generally be operated according tothe manufacturer's instructions.

Within one aspect of the present invention, use of a HER-2/neupolypeptide (or a DNA molecule that directs the expression of such apeptide) to generate an immune response to the HER-2/neu protein(including that expressed on a malignancy in which a HER-2/neu oncogeneis associated) may be detected. Representative examples of suchmalignancies include breast, ovarian, colon, lung and prostate cancers.An immune response to the HER-2/neu protein, once generated by aHER-2/neu polypeptide, can be long-lived and can be detected long afterimmunization, regardless of whether the protein is present or absent inthe body at the time of testing. An immune response to the HER-2/neuprotein generated by reaction to a HER-2/neu polypeptide can be detectedby examining for the presence or absence, or enhancement, of specificactivation of CD4⁺ or CD8⁺ T cells. More specifically, T cells isolatedfrom an immunized individual by routine techniques (such as byFicoll/Hypaque density gradient centrifugation of peripheral bloodlymphocytes) are incubated with HER-2/neu protein. For example, T cellsmay be incubated in vitro for 2-9 days (typically 4 days) at 37° C. withHER-2/neu protein (typically, 5 μg/ml of whole protein or graded numbersof cells synthesizing HER-2/neu protein). It may be desirable toincubate another aliquot of a T cell sample in the absence of HER-2/neuprotein to serve as a control.

Specific activation of CD4⁺ or CD8⁺ T cells may be detected in a varietyof ways. Methods for detecting specific T cell activation includedetecting the proliferation of T cells, the production of cytokines(e.g., lymphokines), or the generation of cytolytic activity (i.e.,generation of cytotoxic T cells specific for HER-2/neu protein). ForCD4⁺ T cells, a preferred method for detecting specific T cellactivation is the detection of the proliferation of T cells. For CD8⁺ Tcells, a preferred method for detecting specific T cell activation isthe detection of the generation of cytolytic activity.

Detection of the proliferation of T cells may be accomplished by avariety of known techniques. For example, T cell proliferation can bedetected by measuring the rate of DNA synthesis. T cells which have beenstimulated to proliferate exhibit an increased rate of DNA synthesis. Atypical way to measure the rate of DNA synthesis is, for example, bypulse-labeling cultures of T cells with tritiated thymidine, anucleoside precursor which is incorporated into newly synthesized DNA.The amount of tritiated thymidine incorporated can be determined using aliquid scintillation spectrophotometer. Other ways to detect T cellproliferation include measuring increases in interleukin-2 (IL-2)production, Ca²⁺ flux, or dye uptake, such as3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Alternatively,synthesis of lymphokines (such as interferon-gamma) can be measured orthe relative number of T cells that can respond to intactp185^(HER-2/neu) protein may be quantified.

By use or expression of a HER-2/neu polypeptide, T cells which recognizethe HER-2/neu protein can be proliferated in vivo. For example,immunization with a HER-2/neu peptide (i.e., as a vaccine) can inducecontinued expansion in the number of T cells necessary for therapeuticattack against a tumor in which the HER-2/neu oncogene is associated.Typically, about 0.01 μg/kg to about 100 mg/kg body weight will beadministered by the intradermal, subcutaneous or intravenous route. Apreferred dosage is about 1 μg/kg to about 1 mg/kg, with about 5 μg/kgto about 200 μg/kg particularly preferred. It will be evident to thoseskilled in the art that the number and frequency of administration willbe dependent upon the response of the patient. It may be desirable toadminister the HER-2/neu polypeptide repetitively. It will be evident tothose skilled in this art that more than one HER-2/neu polypeptide maybe administered, either simultaneously or sequentially. Preferredpeptides for immunization are those that include the amino acid sequenceof SEQ ID NO:2 beginning at about the lysine residue at amino acidposition 676 and extending to about the valine residue at amino acidposition 1255. It will be appreciated by those in the art that thepresent invention contemplates the use of an intact HER-2/neupolypeptide as well as division of such a polypeptide into a pluralityof peptides. Neither intact p185^(HER-2/neu) protein nor a peptidehaving the amino acid sequence of its entire extracellular domain (i.e.,a peptide having an amino acid sequence of SEQ ID NO:2 from amino acidposition 1 up to amino acid position 650, plus or minus about one tofive positions, and with or without the first 21 amino acid positions)are used alone for immunization.

A HER-2/neu polypeptide (or nucleic acid) is preferably formulated foruse in the above methods as a pharmaceutical composition (e.g.,vaccine). Pharmaceutical compositions generally comprise one or morepolypeptides in combination with a pharmaceutically acceptable carrier,excipient or diluent. Such carriers will be nontoxic to recipients atthe dosages and concentrations employed. The use of a HER-2/neupolypeptide in conjunction with chemotherapeutic agents is alsocontemplated.

In addition to the HER-2/neu polypeptide (which functions as anantigen), it may be desirable to include other components in thevaccine, such as a vehicle for antigen delivery and immunostimulatorysubstances designed to enhance the protein's immunogenicity. Examples ofvehicles for antigen delivery include aluminum salts, water-in-oilemulsions, biodegradable oil vehicles, oil-in-water emulsions,biodegradable microcapsules, and liposomes. Examples ofimmunostimulatory substances (adjuvants) includeN-acetylmuramyl-L-alanine-D-isoglutamine (MDP), lipopoly-saccharides(LPS), glucan, IL-12, GM-CSF, gamma interferon and IL-15. It will beevident to those of ordinary skill in this art that a HER-2/neupolypeptide for a vaccine may be prepared synthetically or be naturallyderived.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administrationand whether a sustained release is desired. For parenteraladministration, such as subcutaneous injection, the carrier preferablycomprises water, saline, alcohol, a fat, a wax or a buffer. For oraladministration, any of the above carriers or a solid carrier, such asmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, sucrose, and magnesium carbonate, may beemployed. Biodegradable microspheres (e.g., polylactic galactide) mayalso be employed as carriers for the pharmaceutical compositions of thisinvention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109. A HER-2/neupolypeptide may be encapsulated within the biodegradable microsphere orassociated with the surface of the microsphere. For example, in apreferred embodiment, a polypeptide having the amino acid sequence ofSEQ ID NO:2 from amino acid 676 through amino acid 1255 is encapsulatedwithin a biodegradable microsphere. In this regard, it is preferablethat the microsphere be larger than approximately 25 microns.

Pharmaceutical compositions (including vaccines) may also containdiluents such as buffers, antioxidants such as ascorbic acid, lowmolecular weight (less than about 10 residues) polypeptides, proteins,amino acids, carbohydrates including glucose, sucrose or dextrins,chelating agents such as EDTA, glutathione and other stabilizers andexcipients. Neutral buffered saline or saline mixed with nonspecificserum albumin are exemplary appropriate diluents. Preferably, product isformulated as a lyophilizate using appropriate excipient solutions(e.g., sucrose) as diluents.

As an alternative to the presentation of HER-2/neu polypeptides, thesubject invention includes compositions capable of delivering nucleicacid molecules encoding a HER-2/neu polypeptide. Such compositionsinclude recombinant viral vectors (e.g., retroviruses (see WO 90/07936,WO 91/02805, WO 93/25234, WO 93/25698, and WO 94/03622), adenovirus (seeBerkner, Biotechniques 6:616-627, 1988; Li et al., Hum. Gene Ther.4:403-409, 1993; Vincent et al., Nat. Genet. 5:130-134, 1993; and Kollset al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994), pox virus (seeU.S. Pat. No. 4,769,330; U.S. Pat. No. 5,017,487; and WO 89/01973)),naked DNA (see WO 90/11092), nucleic acid molecule complexed to apolycationic molecule (see WO 93/03709), and nucleic acid associatedwith liposomes (see Wang et al., Proc. Natl. Acad. Sci. USA 84:7851,1987). In certain embodiments, the DNA may be linked to killed orinactivated adenovirus (see Curiel et al., Hum. Gene Ther. 3:147-154,1992; Cotton et al., Proc. Natl. Acad. Sci. USA 89:6094, 1992). Othersuitable compositions include DNA-ligand (see Wu et al., J. Biol. Chem.264:16985-16987, 1989) and lipid-DNA combinations (see Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-7417, 1989). In addition, theefficiency of naked DNA uptake into cells may be increased by coatingthe DNA onto biodegradable beads.

In addition to direct in vivo procedures, ex vivo procedures may be usedin which cells are removed from an animal, modified, and placed into thesame or another animal. It will be evident that one can utilize any ofthe compositions noted above for introduction of HER-2/neu nucleic acidmolecules into tissue cells in an ex vivo context. Protocols for viral,physical and chemical methods of uptake are well known in the art.

Accordingly, the present invention is useful for enhancing or eliciting,in a patient or cell culture, a cellular immune response (e.g., thegeneration of antigen-specific cytolytic T cells). As used herein, theterm “patient” refers to any warm-blooded animal, preferably a human. Apatient may be afflicted with cancer, such as breast cancer, or may benormal (i.e., free of detectable disease and infection). A “cellculture” is any preparation of T cells or isolated component cells(including, but not limited to, macrophages, monocytes, B cells anddendritic cells). Such cells may be isolated by any of a variety oftechniques well known to those of ordinary skill in the art (such asFicoll-hypaque density centrifugation). The cells may (but need not)have been isolated from a patient afflicted with a HER-2/neu associatedmalignancy, and may be reintroduced into a patient after treatment.

The present invention also discloses that HER-2/neu polypeptide, inaddition to being immunogenic to T cells, appears to stimulate B-cellsto produce antibodies capable of recognizing HER-2/neu polypeptide.Antibodies specific (i.e., which exhibit a binding affinity of about 10⁷liters/mole or better) for HER-2/neu protein may be found in a varietyof body fluids including sera and ascites. Briefly, a body fluid sampleis isolated from a warm-blooded animal, such as a human, for whom it isdesired to determine whether antibodies specific for HER-2/neupolypeptide are present. The body fluid is incubated with HER-2/neupolypeptide under conditions and for a time sufficient to permitimmunccomplexes to form between the polypeptide and antibodies specificfor the protein. For example, a body fluid and HER-2/neu polypeptide maybe incubated at 4° C. for 24-48 hours. Following the incubation, thereaction mixture is tested for the presence of immunocomplexes.Detection of one or more immunocomplexes formed between HER-2/neupolypeptide and antibodies specific for HER-2/neu polypeptide may beaccomplished by a variety of known techniques, such as radioimmunoassays(RIA) and enzyme linked immunosorbent assays (ELISA).

Suitable immunoassays include the double monoclonal antibody sandwichimmunoassay technique of David et al. (U.S. Pat. No. 4,376,110);monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkhamand Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone,Edinburgh, 1970); the “western blot” method of Gordon et al. (U.S. Pat.No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al., J.Biol. Chem. 255:4980-4983, 1980); enzyme-linked immunosorbent assays asdescribed by, for example, Raines and Ross (J. Biol. Chem.257:5154-5160, 1982); immunocytochemical techniques, including the useof fluorochromes (Brooks et al., Clin. Exp. Immunol. 39: 477, 1980); andneutralization of activity [Bowen-Pope et al., Proc. Natl. Acad. Sci.USA 81:2396-2400 (1984)], all of which are hereby incorporated byreference. In addition to the immunoassays described above, a number ofother immunoassays are available, including those described in U.S. Pat.Nos. 3,817,827; 3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; and 4,098,876, all of which are herein incorporated byreference.

For detection purposes, HER-2/neu polypeptide (“antigen”) may either belabeled or unlabeled. When unlabeled, the antigen finds use inagglutination assays. In addition, unlabeled antigen can be used incombination with labeled molecules that are reactive withimmunocomplexes, or in combination with labeled antibodies (secondantibodies) that are reactive with the antibody directed againstHER-2/neu polypeptide, such as antibodies specific for immunoglobulin.Alternatively, the antigen can be directly labeled. Where it is labeled,the reporter group can include radioisotopes, fluorophores, enzymes,luminescers, or dye particles. These and other labels are well known inthe art and are described, for example, in the following U.S. patents:U.S. Pat. Nos. 3,766,162; 3,791,932; 3,817,837; 3,996,345; and4,233,402.

Typically in an ELISA assay, antigen is adsorbed to the surface of amicrotiter well. Residual protein-binding sites on the surface are thenblocked with an appropriate agent, such as bovine serum albumin (BSA),heat-inactivated normal goat serum (NGS), or BLOTTO (buffered solutionof nonfat dry milk which also contains a preservative, salts, and anantifoaming agent). The well is then incubated with a sample suspectedof containing specific antibody. The sample can be applied neat, or,more often, it can be diluted, usually in a buffered solution whichcontains a small amount (0.1%-5.0% by weight) of protein, such as BSA,NGS, or BLOTTO. After incubating for a sufficient length of time toallow specific binding to occur, the well is washed to remove unboundprotein and then incubated with an anti-species specific immunoglobulinantibody labeled with a reporter group. The reporter group can be chosenfrom a variety of enzymes, including horseradish peroxidase,beta-galactosidase, alkaline phosphatase, and glucose oxidase.Sufficient time is allowed for specific binding to occur, then the wellis again washed to remove unbound conjugate, and the substrate for theenzyme is added. Color is allowed to develop and the optical density ofthe contents of the well is determined visually or instrumentally.

In one preferred embodiment of this aspect of the present invention, areporter group is bound to HER-2/neu protein. The step of detectingimmunocomplexes involves removing substantially any unbound HER-2/neuprotein and then detecting the presence or absence of the reportergroup.

In another preferred embodiment, a reporter group is bound to a secondantibody capable of binding to the antibodies specific for HER-2/neuprotein. The step of detecting immunocomplexes involves (a) removingsubstantially any unbound antibody, (b) adding the second antibody, (c)removing substantially any unbound second antibody and then (d)detecting the presence or absence of the reporter group. Where theantibody specific for HER-2/neu protein is derived from a human, thesecond antibody is an anti-human antibody.

In a third preferred embodiment for detecting immunocomplexes, areporter group is bound to a molecule capable of binding to theimmunocomplexes. The step of detecting involves (a) adding the molecule,(b) removing substantially any unbound molecule, and then (c) detectingthe presence or absence of the reporter group. An example of a moleculecapable of binding to the immunocomplexes is protein A.

It will be evident to one skilled in the art that a variety of methodsfor detecting the immunocomplexes may be employed within the presentinvention. Reporter groups suitable for use in any of the methodsinclude radioisotopes, fluorophores, enzymes, luminescers, and dyeparticles.

In a related aspect of the present invention, detection ofimmunocomplexes formed between HER-2/neu polypeptide and antibodies inbody fluid which are specific for HER-2/neu polypeptide may be used tomonitor the effectiveness of cancer therapy, which involves a HER-2/neupolypeptide, for a malignancy in which the HER-2/neu oncogene isassociated. Samples of body fluid taken from an individual prior to andsubsequent to initiation of therapy may be analyzed for theimmunocomplexes by the methodologies described above. Briefly, thenumber of immunocomplexes detected in both samples are compared. Asubstantial change in the number of immunocomplexes in the second sample(post-therapy initiation) relative to the first sample (pre-therapy)reflects successful therapy.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Expression and Purification of Recombinant HumanHER-2/Neu Polypeptide

The human HER-2/neu polypeptide was recovered by the PCR method (e.g.,U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159) from a plasmid preparedaccording to Di Fiore et al. (King et al., Science 229:974-976, 1985; DiFiore et al., Science 237:178-182, 1987) using oligonucleotide primersthat additionally introduced a BssHII restriction site and anenterokinase protease site on the 5′ end and an EcoRI site on the 3′end. The primer for the 5′-end was5′-TCTGGCGCGCTGGATGACGATGACAAGAAACGACGGCAGCAGAAGATC-3′ (SEQ ID NO:3)while the primer for the 3′-end was5′-TGAATTCTCGAGTCATTACACTGGCACGTCCAGACCCAG-3′ (SEQ ID NO:4). Theresulting 1.8 kb PCR fragment was subcloned into the T-vector fromNovagen (Madison, Wis., USA) and the sequence of selected clones wasdetermined on the ABI 373 automated DNA sequencer (Applied BiosystemsInc., Foster City, Calif., USA) using overlapping sequencing primers.PCR fragments with sequence that corresponded to the published DNAsequence for the human HER-2/neu cDNA (SEQ ID NO:1; Coussens et al.,Science 230:1132, 1985; Yamamoto et al., Nature 319:230, 1986) were thenconnected in the correct reading frame via the BssHII site to a modifiedE. coli thioredoxin reductase. A 6× histidine affinity tag employed inNi-NTA affinity purification of the expressed fusion protein wasincorporated into the thioredoxin reductase fusion partner. This cDNAfor the trxA-human HER-2/neu polypeptide fusion protein was subclonedinto a modified pET expression vector for expression in E. coli.

While thioredoxin reductase has been reported to stabilize andsolubilize other heterologous proteins expressed in E. coli, it did notappear to offer any significant advantage for human HER-2/neupolypeptide expression in E. coli. While a significant proportion of thetrxA-HER-2/neu polypeptide fusion protein was soluble, a majority wasexpressed in inclusion bodies. The fusion protein was also subjected todegradation du ring expression in E. coli. The presence of thethioredoxin reductase fusion partner may, however, stabilize the proteinduring purification. The availability of monoclonal antibodies tothioredoxin reductase provides a convenient marker to follow duringpurification.

For purification of the human HER-2/neu polypeptide with the thioredoxinreductase fusion partner containing the 6×His affinity tag, the E. colipellet was resuspended with protease inhibitors and lysozyme andsonicated. The inclusion bodies were isolated by centrifugation, and arewashed 3× with deoxycholate, the last wash being overnight to removeLPS. The washed inclusion bodies are solubilized in GuHCl for Nipurification. The Ni column was eluted with Imidazole in urea anddialyzed against 10 mM Tris pH8. The recovery of HER-2/neu polypeptideusing this protocol was from 80%-95% pure full length protein with themain contaminant being degraded protein. From 500 ml of fermentation, 20mg were recovered. It was >98% HER-2/neu polypeptide. The techniquesused herein are well known to those in the art and have been described,for example, in J. Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989, Cold SpringHarbor, N.Y., USA.

Example 2 Dendritic Cells can Prime Human HER-2/Neu Polypeptide

A. Generation of DC Cultures from Bone Marrow

DC cultures were generated from CD34+ hematopoietic progenitor cells(HPC). CD34+ cells were purified from bone marrow of normal donors usingthe cell separation system Ceprate LC Kit (CellPro, Bothell, Wash.,USA). Purity of recovered CD34+ cells was determined by flow cytometricanalysis to be 80% to 95%. CD34+ cells were cultured in serum-freemedium (X-VIVO 10, Biowhittaker, Inc., Walkersville, Md., USA)supplemented with L-glutamine (584 μg/l), penicillin (10 IU/ml),streptomycin (100 μg/ml), 100 ng/ml human rGM-CSF and 50 ng/ml humanrIL-6 (Immunex, Seattle, Wash., USA). After 0 to 17 days of culturetime, cells were harvested and used for phenotyping and T cellstimulation assays. GM-CSF alone and in combination with IL-4 or TNFαhave been described to induce the in vitro growth of DC. In experimentsusing KLH and OVA as antigens to prime naive T cells, GM-CSF plus IL-6consistently gave a comparable total stimulation, but with a lowerbackground and thus a higher stimulation index as compared to GM-CSFplus IL-4 or TNFα.

B. T Cell Priming Assay

Bone marrow derived CD34+ HPC cultured in serum-free medium containingGM-CSF and IL-6 were used as APC after a culture period of 0-17 days.Priming ability of DC was determined by culturing them with autologous,naive T lymphocytes in the presence or absence of the protein antigenrecombinant human HER-2/neu polypeptide (hHNP) (10 μg/ml). CD4+ Tlymphocytes were isolated from peripheral blood mononuclear cells bypositive selection using immunoaffinity columns (CellPro, Inc., Bothell,Wash., USA). CD4+CD45RA+ (naive) T lymphocytes were selected from CD4+ Tlymphocytes using an anti-CD45RA mAb directly conjugated to FITC(Immunotech, Westbrook, Me., USA) by flow cytometric sorting. TheCD4+CD45RA+ T cells obtained were 99% pure. DC cultures were plated into96-well round-bottomed plates (Corning, Corning, N.Y., USA) at variousconcentrations and incubated for 16-18 hours with hHNP 10 μg/ml finalconcentration. Antigen-pulsed DC were irradiated (10 Gy), and autologousCD4+CD45RA+ T lymphocytes were added (5×10⁴/well). Proliferativeresponse of T cells was measured by the uptake of (³H)thymidine (1μCi/well) added on day 6 for 16-18 hours. Proliferation assays wereperformed in serum-free and cytokine-free medium. The results are shownin FIG. 1. FIG. 2 shows the results of testing CD4+ T cells, from anormal donor, for responses to hHNP. Similar data was obtained with Tcells from nine out of ten normal individuals.

Example 3 Assay for Detecting Low Frequency Lymphocyte Precursors

Three assays can be used for the detection of CD4⁺ responses: a standardproliferation assay, a screening method for low frequency events, and alimiting dilution assay (LDA). Conventional proliferative assays arecapable of readily detecting primed responses. The proliferativeresponse stimulation index provides a rough correlation with precursorfrequency of antigen-reactive cells. Any specific proliferative responsedetected from PBL is considered to be a primed response.

To provide a more quantitative interpretation of CD4⁺ T cell responses,the assay system developed for detecting low lymphocyte precursorfrequency responses (described below) is used. This assay is simple andcost-effective. In circumstances in which more precision is needed, theprecursor frequency is validated by limiting dilution assays (Bishop andOrosz, Transplantation 47:671-677, 1989).

Responses greater than detected in normal individuals are defined as aprimed response and imply existent immunity. Low responses, detectableonly by LDA conditions are considered to be unprimed responses. Anabsent response by LDA or a response lower than that defined by thenormal population analysis is considered to be tolerance/anergy.

In general, primed CD4⁺ T cell responses can be detected in conventionalproliferative assays, whereas unprimed responses are not detectable inthe same assays.

Detection of small numbers of unprimed T cells is limited by confoundingbackground thymidine uptake including the autologous mixed lymphocyteresponse (AMLR) to self MHC antigen plus responses to processed selfserum proteins and exogenously added serum proteins.

To elicit and detect unprimed T cells, an assay system for low frequencyresponses based on Poisson sampling statistics was used (In: Pinnacles,Chiron Corporation, 1:1-2, 1991). This type of analysis appliesspecifically to low frequency events in that, if the precursor frequencyis less than the number of cells in one replicate culture, manyreplicates are required to detect a statistically significant number ofpositives.

Theoretically, the analysis will correct for autologous responses bysetting up a known positive control (such as PHA or tetanus toxoid) andknown negative control (no antigen) and evaluating all data points fromlowest to highest irrespective of the experimental group to which theybelong. A cutoff value is calculated based on the equationcutoff=M+(F+SD), where M=arithmetic mean, F=3.29, a factor from tablesof standardized normal distribution chosen so not more than 0.1% of the“true negatives” of a normally distributed background will be above thecutoff, and SD=standard deviation. In this screening assay, wells abovethe cutoff are considered true positives that potentially contain alymphocyte that is specifically proliferating to the antigen ofinterest. Although estimations of lymphocyte precursor frequency ispossible using this method, precise determination requires formal LDAanalysis.

Example 4 HER-2/Neu Polypeptide Based Vaccine Elicits Immunity toHER-2/Neu Protein

A. Animals

Rats used in this study were Fischer strain 344 (CDF (F-344)/CrlBR)(Charles River Laboratories, Portage Mich.). Animals were maintained atthe University of Washington Animal facilities under specific pathogenfree conditions and routinely used for experimental studies between 3and 4 months of age.

B. Immunization

Fischer rats were immunized with recombinant rat HER-2/neu polypeptide(rHNP) in a variety of adjuvants (MPL, Vaccel; Ribi, Bozeman, Mont.,USA). Animals received 50 μg of rHNP mixed with adjuvant subcutaneously.Twenty days later the animals were boosted with a second immunization of50 μg of rHNP administered in the same fashion. Twenty days after thebooster immunization animals were tested for the presence of antibodiesdirected against rat HER-2/neu protein (neu).

C. Cell Lines

Two cell lines were used as a source of neu proteins. SKBR3, a humanbreast cancer cell line that is a marked overexpressor of HER-2/neu(American Type Cuiture Collection, Rockville, Md.), was maintained inculture in 10% fetal bovine serum (FBS) (Gemini Bioproducts, Inc.,Calabasas, Calif.) and RPMI. DHFR-G8, an NIH/3T3 cell line cotransfectedwith cneu-p and pSV2-DHFR (American Type Culture Collection, Rockville,Md.), was used as a source of non-transforming rat neu protein (Bernardset al., Proc. Natl. Acad. Sci. USA 84:6854-6858, 1987). This cell linewas maintained in 10% FBS and Dulbecco's modified Eagle's medium with4.5 g/L glucose. DHFR-G8 cells were passaged through the same mediumsupplemented with 0.3 μM methotrexate at every third passage to maintainthe neu transfectant.

D. Preparation of Cell Lysates

Lysates of both SKBR3 and DHFR-G8 were prepared and used as a source ofneu protein. Briefly, a lysis buffer consisting of tris base, sodiumchloride and Triton-X (1%) pH 7.5 was prepared. Protease inhibitors wereadded; aprotinin (1 μg/ml), benzamidine (1 mM) and PMSF (1 mM). 1 ml ofthe lysis buffer was used to suspend 10⁷ cells. The cells were vortexedfor 15 seconds every 10 minutes for an hour until disrupted. Allprocedures were performed on ice in a 4° C. cold room. After disruptionthe cells were microfuged at 4° C. for 20 minutes. Supernatant wasremoved from cell debris and stored in small aliquots at −70° C. untilused. Presence of human and rat neu in the lysates was documented byWestern blot analysis.

E. ELISA for Rat Neu Antibody Responses

96 well Immulon 4 plates (Baxter SP, Redmond, Wash.: DynatechLaboratories) were incubated overnight at 4° C. with a rat neu specificmonoclonal antibody (Oncogene Science), 7.16.4, at a concentration of 10μg/ml diluted in carbonate buffer (equimolar concentrations of Na₂CO₃and NaHCO₃ pH 9.6). After incubation, all wells were blocked with PBS-1%BSA (Sigma Chemical, St. Louis, Mo., USA), 100 μl/well for 3 hours atroom temperature. The plate was washed with PBS-0.5% Tween and lysatesof DHFRG8, a murine cell line transfected with rat neu DNA (American.Type Culture Collection, Rockville, Md., USA); a source of rat neuprotein, were added to alternating rows. The plate was incubatedovernight at 4° C. The plate was then washed with PBS-0.5% Tween andexperimental sera was added at the following dilutions: 1:25 to 1:200.The sera was diluted in PBS-1% BSA-1% FBS-25 μg/ml mouse IgG-0.01% NaN₃and then serially into PBS-1% BSA. 50 μl of diluted sera was added/welland incubated 1 hour at room temperature. Each experimental sera wasadded to a well with rat neu and a well without rat neu. Sheep anti-ratIg F(ab′)₂ horseradish peroxidase (HRP) was added to the wells at a1:5000 dilution in PBS-1% BSA and incubated for 45 minutes at roomtemperature (Amersham Co., Arlington Heights, Ill., USA). Following thefinal wash, TMB (Kirkegaard and Perry Laboratories, Gaithersburg, Md.)developing reagent was added. Color reaction was read at an opticaldensity of 450 nm. The OD of each serum dilution was calculated as theOD of the rat neu coated wells minus the OD of the PBS-1% BSA coatedwells. Sera from animals immunized with the adjuvants alone and ananimal immunized with hHNP (foreign protein) were also evaluated in asimilar manner. The results are shown in FIG. 3.

F. T Cell Proliferation Assays

For analysis of HER-2/neu polypeptide specific responses: Fresh spleenor lymph node cells are harvested by mechanical disruption and passagethrough wire mesh and washed. 2×10⁵ spleen cells/well and 1×10⁵ lymphnode cells/well are plated into 96-well round bottom microtiter plates(Corning, Corning, N.Y.) with 6 replicates per experimental group. Themedia consists of EHAA 120 (Biofluids) with L-glutamine,penicillin/streptomycin, 2-mercaptoethanol, and 5% FBS. Cells areincubated with polypeptides. After 4 days, wells are pulsed with 1 μCiof [³H]thymidine for 6-8 hours and counted. Data is expressed as astimulation index (SI) which is defined as the mean of the experimentalwells divided by the mean of the control wells (no antigen). Foranalysis of HER-2/neu protein specific responses: Spleen or lymph nodecells are cultured for 3 in vitro stimulations. At the time of analysis1×10⁵ cultured spleen or lymph node T cells are plated into 96 wellmicrotiter plates as described above. Cells are incubated with 1 μg/mlimmunoaffinity column purified rat neu (from DHFR-G8 cells as the sourceof rat neu). After 4 days, wells were pulsed with 1 μCi of [³H]thymidinefor 6-8 hours and counted. Data is expressed as a stimulation indexwhich is defined as the mean of the experimental wells divided by themean of the control wells (no antigen).

Example 5 Primed Responses to Human HER-2/Neu Polypeptide Can beDetected in Patients with Breast Cancer

Heparinized blood was obtained from a patient with stage II HER-2/neuoverexpressing breast cancer. Peripheral blood mononuclear cells (PBMC)were separated by Ficoll Hypaque density centrifugation. PBMC wereplated at a concentration of 2×10⁵/well into 96-well round-bottomedplates (Corning, Corning, N.Y., USA). 24 well replicates were performedfor each experimental group. Antigens consisting of HER-2/neu derivedpeptides (15-20 amino acids in length with number of first amino acid insequence listed) 25 μg/ml, human HER-2/neu polypeptide (hHNP) 1 μg/ml,tetanus toxoid 1 μg/ml, and p30 a peptide derived from tetanus 25 μg/mlwere added to each 24 well replicate. The assay was performed in mediacontaining 10% human sera. Proliferative response of T cells wasmeasured by the uptake of (³H)thymidine (1 μCi/well) added on day 4 for10 hours. Positive wells, antigen reactive wells, were scored aspositive if the cpm was greater than the mean and 3 standard deviationsof the no antigen wells. The results are shown in FIG. 4. This stage IIbreast cancer patient has a significant response to recombinant hHNP.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually incorporated by reference.

From the foregoing, it will be evident that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1. A polypeptide encoded by a DNA sequence selected from: (a)nucleotides 2026 through 3765 of SEQ ID NO:1; and (b) DNA sequences thathybridize to a nucleotide sequence complementary to nucleotides 2026through 3765 of SEQ ID NO:1 under moderately stringent conditions,wherein the DNA sequence encodes a polypeptide that produces an immuneresponse to HER-2/neu protein.
 2. A polypeptide having the amino acidsequence of SEQ ID NO:2 from lysine, amino-acid 676, through valine,amino acid 1255, or a variant thereof that produces at least anequivalent immune response.
 3. A polypeptide according to claim 2 havingthe amino acid sequence of SEQ ID NO:2 from amino acid 676 through aminoacid
 1255. 4. A composition comprising a polypeptide according to anyone of claims 1, 2 or 3, in combination with a pharmaceuticallyacceptable carrier or diluent.
 5. A nucleic acid molecule directing theexpression of a polypeptide according to any one of claims 1, 2 or
 3. 6.A viral vector directing the expression of a polypeptide according toany one of claims 1, 2 or
 3. 7. A method for eliciting or enhancing animmune response to HER-2/neu protein, comprising administering to awarm-blooded animal in an amount effective to elicit or enhance saidresponse a polypeptide according to any one of claims 1, 2 or 3, or anucleic acid molecule according to claim 5, or a viral vector accordingto claim
 6. 8. A method according to claim 7 wherein the step ofadministering comprises transfecting cells of the animal ex vivo withthe nucleic acid molecule and subsequently delivering the transfectedcells to the animal.
 9. A method according to claim 7 wherein the stepof administering comprises infecting cells of the animal ex vivo withthe viral vector and subsequently delivering the infected cells to theanimal.